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Bacillus velezensis has gained increasing recognition as a probiotic for improving animal growth performance and gut health. We identified six B. velezensis strains from sixty Bacillus isolates that were isolated from the cecal samples of fifteen different chicken breeds. We characterized the probiotic properties of these six B. velezensis strains. The effect of a selected strain (B. velezensis CML532) on chicken growth performance under normal feeding and Clostridium perfringens challenge conditions was also evaluated. The results revealed that the six B. velezensis strains differed in their probiotic properties, with strain CML532 exhibiting the highest bile salt and acid tolerance and high-yield enzyme and antibacterial activities. Genomic analyses showed that genes related to amino acid and carbohydrate metabolism, as well as genes related to starch and cellulose hydrolysis, were abundant in strain CML532. Dietary supplementation with strain CML532 promoted chicken growth, improved the gut barrier and absorption function, and modulated the gut microbiota. Under the C. perfringens challenge condition, strain CML532 alleviated intestinal damage, reduced ileal colonization of C. perfringens, and also improved chicken growth performance. Collectively, this study demonstrated that the newly isolated B. velezensis strain is a promising probiotic with beneficial effects on chicken growth performance and gut health. [ABSTRACT FROM AUTHOR]